Abstract
In this study, we compared three different methods used for quantification of gene electrotransfer efficiency: fluorescence microscopy, flow cytometry and spectrofluorometry. We used CHO and B16 cells in a suspension and plasmid coding for GFP. The aim of this study was to compare and analyse the results obtained by fluorescence microscopy, flow cytometry and spectrofluorometry and in addition to analyse the applicability of spectrofluorometry for quantifying gene electrotransfer on cells in a suspension. Our results show that all the three methods detected similar critical electric field strength, around 0.55 kV/cm for both cell lines. Moreover, results obtained on CHO cells showed that the total fluorescence intensity and percentage of transfection exhibit similar increase in response to increase electric field strength for all the three methods. For B16 cells, there was a good correlation at low electric field strengths, but at high field strengths, flow cytometer results deviated from results obtained by fluorescence microscope and spectrofluorometer. Our study showed that all the three methods detected similar critical electric field strengths and high correlations of results were obtained except for B16 cells at high electric field strengths. The results also demonstrated that flow cytometry measures higher values of percentage transfection compared to microscopy. Furthermore, we have demonstrated that spectrofluorometry can be used as a simple and consistent method to determine gene electrotransfer efficiency on cells in a suspension.
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Belehradek M, Domenge C, Luboinski B et al (1993) Electrochemotherapy, a new antitumor treatment. First clinical phase I-II trial. Cancer 72:3694–3700. doi:10.1002/1097-0142(19931215)72:12<3694:AID-CNCR2820721222>3.0.CO;2-2
Bureau MF, Gehl J, Deleuze V et al (2000) Importance of association between permeabilization and electrophoretic forces for intramuscular DNA electrotransfer. Biochim Biophys Acta 1474:353–359. doi:10.1016/S0304-4165(00)00028-3
Černe K, Erman A, Veranič P (2013) Analysis of cytotoxicity of melittin on adherent culture of human endothelial cells reveals advantage of fluorescence microscopy over flow cytometry and haemocytometer assay. Protoplasma 2013:1–7. doi:10.1007/s00709-013-0489-8
Daud AI, DeConti RC, Andrews S et al (2008) Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma. J Clin Oncol 26:5896–5903. doi:10.1200/JCO.2007.15.6794
Degelau A, Scheper T, Bailey JE, Guske C (1995) Fluorometric measurement of poly-β hydroxybutyrate in Alcaligenes eutrophus by flow cytometry and spectrofluorometry. Appl Microbiol Biotechnol 42:653–657. doi:10.1007/BF00171939
Faurie C, Phez E, Golzio M et al (2004) Effect of electric field vectoriality on electrically mediated gene delivery in mammalian cells. Biochim Biophys Acta 1665:92–100. doi:10.1016/j.bbamem.2004.06.018
Faurie C, Rebersek M, Golzio M et al (2010) Electro-mediated gene transfer and expression are controlled by the life-time of DNA/membrane complex formation. J Gene Med 12:117–125. doi:10.1002/jgm.1414
Favard C, Dean D, Rols M (2007) Electrotransfer as a non viral method of gene delivery. Curr Gene Ther 7:67–77
Gabrijel M, Repnik U, Kreft M et al (2004) Quantification of cell hybridoma yields with confocal microscopy and flow cytometry. Biochem Biophys Res Commun 314:717–723. doi:10.1016/j.bbrc.2003.12.154
Gehl J, Sørensen TH, Nielsen K et al (1999) In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution. Biochim Biophys Acta 1428:233–240. doi:10.1016/S0304-4165(99)00094-X
Golzio M, Teissié J, Rols M-P (2001) Control by membrane order of voltage-induced permeabilization, loading and gene transfer in mammalian cells. Bioelectrochemistry 53:25–34. doi:10.1016/S0302-4598(00)00091-X
Golzio M, Teissié J, Rols M-P (2002) Direct visualization at the single-cell level of electrically mediated gene delivery. PNAS 99:1292–1297. doi:10.1073/pnas.022646499
Golzio M, Rols MP, Teissié J (2004) In vitro and in vivo electric field-mediated permeabilization, gene transfer, and expression. Methods 33:126–135. doi:10.1016/j.ymeth.2003.11.003
Haberl S, Miklavcic D, Pavlin M (2010) Effect of Mg ions on efficiency of gene electrotransfer and on cell electropermeabilization. Bioelectrochemistry 79:265–271. doi:10.1016/j.bioelechem.2010.04.001
Haberl S, Kandušer M, Flisar K et al (2013) Effect of different parameters used for in vitro gene electrotransfer on gene expression efficiency, cell viability and visualization of plasmid DNA at the membrane level. J Gene Med 15:169–181. doi:10.1002/jgm.2706
Heller R, Jaroszeski M, Atkin A et al (1996) In vivo gene electroinjection and expression in rat liver. FEBS Lett 389:225–228. doi:10.1016/0014-5793(96)00590-X
Heller R, Gilbert R, Jaroszeski MJ (1999) Clinical applications of electrochemotherapy. Adv Drug Deliv Rev 35:119–129. doi:10.1016/S0169-409X(98)00067-2
Hojman P, Zibert J, Gissel H et al (2007) Gene expression profiles in skeletal muscle after gene electrotransfer. BMC Mol Biol 8:56. doi:10.1186/1471-2199-8-56
Kanduser M, Miklavcic D, Pavlin M (2009) Mechanisms involved in gene electrotransfer using high- and low-voltage pulses—An in vitro study. Bioelectrochemistry 74:265–271. doi:10.1016/j.bioelechem.2008.09.002
Mali B, Jarm T, Snoj M et al (2013) Antitumor effectiveness of electrochemotherapy: a systematic review and meta-analysis. Eur J Surg Oncol 39:4–16. doi:10.1016/j.ejso.2012.08.016
Marjanovic I, Haberl S, Miklavcic D et al (2010) Analysis and comparison of electrical pulse parameters for gene electrotransfer of two different cell lines. J Membr Biol 236:97–105. doi:10.1007/s00232-010-9282-1
Marty M, Sersa G, Garbay JR et al (2006) Electrochemotherapy—an easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study. Eur J Cancer 4:3–13. doi:10.1016/j.ejcsup.2006.08.002
Mir LM, Orlowski S, Belehradek J Jr, Paoletti C (1991) Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses. Eur J Cancer 27:68–72. doi:10.1016/0277-5379(91)90064-K
Mir LM, Bureau MF, Gehl J et al (1999) High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc Natl Acad Sci USA 96:4262–4267
Neumann E, Rosenheck K (1972) Permeability changes induced by electric impulses in vesicular membranes. J Membr Biol 10:279–290. doi:10.1007/BF01867861
Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982) Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1:841–845
Neumann E, Sowers AE, Jordan CA (1989) Electroporation and Electrofusion in Cell Biology. Springer
Paillusson A, Hirschi N, Vallan C et al (2005) A GFP-based reporter system to monitor nonsense-mediated mRNA decay. Nucleic Acids Res 33:e54. doi:10.1093/nar/gni052
Pavlin M, Flisar K, Kanduser M (2010) The role of electrophoresis in gene electrotransfer. J Membr Biol 236:75–79. doi:10.1007/s00232-010-9276-z
Pavlin M, Pucihar G, Kandušer M (2012) The role of electrically stimulated endocytosis in gene electrotransfer. Bioelectrochemistry 83:38–45. doi:10.1016/j.bioelechem.2011.08.005
Puc M, Kotnik T, Mir LM, Miklavčič D (2003) Quantitative model of small molecules uptake after in vitro cell electropermeabilization. Bioelectrochemistry 60:1–10
Rebersek M, Faurie C, Kanduser M et al (2007) Electroporator with automatic change of electric field direction improves gene electrotransfer in vitro. Biomed Eng Online 6:25. doi:10.1186/1475-925X-6-25
Rols M-P, Teissié J (1998) Electropermeabilization of mammalian cells to macromolecules: control by pulse duration. Biophys J 75:1415–1423. doi:10.1016/S0006-3495(98)74060-3
Rosazza C, Escoffre J-M, Zumbusch A, Rols M-P (2011) The actin cytoskeleton has an active role in the electrotransfer of plasmid DNA in mammalian cells. Mol Ther 19:913–921. doi:10.1038/mt.2010.303
Schmid JA, Scholze P, Kudlacek O et al (2001) Oligomerization of the human serotonin transporter and of the rat gaba transporter 1 visualized by fluorescence resonance energy transfer microscopy in living cells. J Biol Chem 276:3805–3810. doi:10.1074/jbc.M007357200
Spanggaard I, Snoj M, Cavalcanti A et al (2013) Gene electrotransfer of plasmid antiangiogenic metargidin peptide (AMEP) in disseminated melanoma: safety and efficacy results of a phase i first-in-man study. Hum Gene Ther Clin Dev 24:99–107. doi:10.1089/humc.2012.240
Sukhorukov VL, Djuzenova CS, Frank H et al (1995) Electropermeabilization and fluorescent tracer exchange: the role of whole-cell capacitance. Cytometry 21:230–240. doi:10.1002/cyto.990210303
Teissié J, Eynard N, Gabriel B, Rols MP (1999) Electropermeabilization of cell membranes. Adv Drug Deliv Rev 35:3–19. doi:10.1016/S0169-409X(98)00060-X
Tesic N, Cemazar M (2013) In vitro targeted gene electrotransfer to endothelial cells with plasmid DNA containing human endothelin-1 promoter. J Membr Biol 246:783–791. doi:10.1007/s00232-013-9548-5
Torrado M, Iglesias R, Mikhailov A (2008) Detection of protein interactions based on GFP fragment complementation by fluorescence microscopy and spectrofluorometry. Biotechniques 44:70–74. doi:10.2144/000112685
Usaj M, Torkar D, Kanduser M, Miklavcic D (2011) Cell counting tool parameters optimization approach for electroporation efficiency determination of attached cells in phase contrast images. J Microsc 2011:304–314. doi:10.1111/j.1365-2818.2010.03441.x
Weaver JC, Chizmadzhev YA (1996) Theory of electroporation: a review. Bioelectrochem Bioenerg 41:135–160. doi:10.1016/S0302-4598(96)05062-3
Wolf H, Rols MP, Boldt E et al (1994) Control by pulse parameters of electric field-mediated gene transfer in mammalian cells. Biophys J 66:524–531. doi:10.1016/S0006-3495(94)80805-7
Acknowledgments
This work was supported by the Slovenian Research Agency within projects: J2-9770, P2-0249, J4-4324, Young Researcher Project and MRIC UL IP-0510 Infrastructure Programme. Authors IM, MK and DM would like to acknowledge that their work has been performed within the scope of LEA EBAM.
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Marjanovič, I., Kandušer, M., Miklavčič, D. et al. Comparison of Flow Cytometry, Fluorescence Microscopy and Spectrofluorometry for Analysis of Gene Electrotransfer Efficiency. J Membrane Biol 247, 1259–1267 (2014). https://doi.org/10.1007/s00232-014-9714-4
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DOI: https://doi.org/10.1007/s00232-014-9714-4